PROJECT SUMMARY The increasing occurrence of antibiotic resistant infections is a major threat to human health, necessitating understanding of mechanisms that confer resistance and development of strategies to counteract them. Antibiotics that bind to the peptidyltransferase center (PTC) of the bacterial ribosome interfere with protein synthesis in bacteria. However, some bacterial strains can modify the PTC region through mutations and post- transcriptional modifications of ribosomal RNA (rRNA), resulting in a ribosome that can no longer bind antibiotics. The multi-drug resistance enzyme Cfr, a member of radical SAM enzyme family, catalyzes methylation of 23S rRNA in the PTC region. This enzyme confers resistance to a number of antibiotics, such as phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A. The ability of Cfr to confer resistance to linezolide, an oxazolidinone antibiotic, is particularly worrisome as this antibiotic is used for the treatment of drug- resistant pathogens including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE). In pathogens, Cfr methylates adenosine A2503 at the C8 position. Interestingly, A2503 is also methylated at its C2 position by RlmN, a radical SAM enzyme that is highly conserved is prokaryotes. C2 A2503 methylation is implicated in the regulation of translational accuracy of the ribosome. A loss of physiological RlmN methylation, both in laboratory selection experiments and in clinical settings, causes antibiotic resistance. These findings suggest that aberrant A2503 methylation ? both the absence of physiological methylation caused by inactivation of RlmN and the hypermethylation caused by acquisition of Cfr ? profoundly impacts susceptibility of the bacterial ribosome to antibiotics. In this application, we will investigate how aberrant methylation of A2503 in 23S rRNA impacts antibiotic resistance and bacterial fitness. Using directed evolution and antibiotic selection, we have evolved variants of RlmN that prevent A2503 methylation and confer resistance to tiamulin. We will determine the molecular basis of the dominant negative effect of RlmN variants. Furthermore, we will investigate how the lack of C2 methylation of A2503 in ribosomes confers antibiotic resistance. Cfr variants, obtained by laboratory evolution or isolated from clinical antibiotic resistant strains, will be used to determine how changes in the sequence of this enzyme modulate methylation of A2503 and how these changes in methylation alter antibiotic susceptibility. We will further assess the impact of aberrant methylation on bacterial fitness and evaluate how changes in methylation influence the regulation of translation. Our work will define how radical SAM-dependent methylation of the PTC regulates the function of the ribosome and modulates its antibiotic susceptibility.